Synaptic vesicle generation from activity-dependent bulk endosomes requires a dephosphorylation-dependent...

Area of Science:

• Neuroscience
• Cell Biology
• Molecular Biology

Background:

• Activity-dependent bulk endocytosis is a key mechanism for synaptic vesicle (SV) regeneration during intense neuronal firing.
• This process involves the formation of bulk endosomes from the plasma membrane, followed by SV generation.
• Calcium efflux and calcineurin activation are known requirements for SV generation from bulk endosomes, but the precise mechanism remains unclear.

Purpose of the Study:

• To elucidate the molecular mechanism by which the protein phosphatase calcineurin mediates synaptic vesicle generation from bulk endosomes.
• To investigate the roles of protein-protein interactions and enzyme activities in this process.

Main Methods:

• Utilized acute interventions in rat primary neuronal cultures to decouple SV generation from bulk endosomes.
• Employed competitive peptides to disrupt protein-protein interactions and specific inhibitors to block enzyme activity.
• Monitored SV generation using morphological (horseradish peroxidase assay) and optical (reserve SV pool replenishment) methods.

Main Results:

• SV generation was inhibited by peptides disrupting calcineurin interactions and by inhibiting dynamin I GTPase activity.
• Disruption of the phosphorylation-dependent dynamin I-syndapin I interaction also inhibited SV generation.
• Localization of calcineurin to bulk endosomes, dynamin I GTPase activity, and the calcineurin-dependent dynamin I-syndapin I interaction were identified as essential for SV generation.

Conclusions:

• Calcineurin acts at bulk endosomes to mediate SV generation.
• Dynamin I GTPase activity is crucial for the fission step in SV generation.
• A calcineurin-dependent dephosphorylation cascade involving dynamin I and syndapin I is essential for generating synaptic vesicles from bulk endosomes.